Ornithine Transcarbamylase Deficiency Wiki
Introduction and Background to Ornithine Transcarbamylase Deficiency Metabolism is the body's process of breaking down substances and building up new materials for its use. As such, it presents a very important aspect of a living organism's ability to thrive and survive. Glycolysis, for example, is the process of breaking down glucose in order to provide energy for the body while the urea cycle is the body's means of managing toxic, nitrogenous wastes from amino acid metabolism. A deficiency of an enzyme in any of these metabolic processes could result in the buildup of metabolic intermediates and compromise the ability to synthesize products required by the body, therefore threatening homeostatic balance and resulting in disease. Due to the multitude of metabolic pathways, there are many opportunities for mutations to affect an organism's well-being. Amongst these many loci of interference, disorders of the urea cycle are one of the most common forms of inherited metabolic diseases. One study in Japan estimates their prevalence to be approximately 1 in 50,000 live births, with ornithine transcarbamylase deficiences (OTCDs) being the most common at 1 in 80,000 births1. Due to its nature as an X-linked recessive disorder, the prevalence of OTCD is comparatively higher in males than females1. This loss of function of an urea cycle enzyme disrupts the metabolism of ammonia and results in hyperammonemia, or a build up of ammonia in the body. The goal of this wiki is to present a comprehensive review of OTCD and outlining some of the literature reporting on the clinical highlights of the disorder, the OTC enzyme’s structure and function, various mutations that may occur within the enzyme, as well as some of the existing management strategies for patients. This page is targeted toward biochemistry students, physicians and researchers, but a brochure is provided at the bottom of the page for patients with OTCD and their families. The Urea Cycle The urea cycle (figure 1) is a crucial metabolic pathway as it is the principal means of excreting the nitrogen that is produced when amino acids are metabolized for energy2. The urea cycle is especially important as the accumulation of ammonia is toxic to the body and has been linked to death as well as various complications, including central nervous system dysfunction and a comatose state. The urea cycle consists of six key enzymes2: # Carbamoyl-phosphate synthetase I (CPS1) # Ornithine transcarbamylase (OTC) # Argininosuccinate synthetase (ASS1) # Argininosuccinate lyase (ASL) # Arginase 1 (ARG1) # N-acetylglutamate synthase (NAGS) The pathway begins in the mitochondria, with N-acetylglutamate being synthesized by NAGS using glutamate and acetyl CoA2. N-acetylglutamate binds to CPS1, which induces a conformational change to reveal the enzyme's active site. CPS1 then catalyzes the synthesis of carbamoyl phosphate by combining a bicarbonate group with an ammonia group formed from protein metabolism. Then, OTC combines the carbamoyl phosphate with ornithine in order to create citrulline2. Citrulline is transported to the cytosol and is combined with asparate by ASS1 in order to make argininosuccinate, which is cleaved by ASL into fumarate and arginine2. Finally ARG1 cleaves arginine to produce ornithine, which returns to the mitochondria via the mitochondrial ornithine transporter 1 protein for re-use in the urea cycle, as well as urea, which is excreted as waste2. In all, this pathway provides the body a means of effectively excreting nitrogen waste, and thus, any disruptions in the urea cycle may result in critical circumstances. Onset of OTC Deficiency and Survival Rate In the 2012 study "Long-term outcome and intervention of urea cycle disorders in Japan” by Kido et al., 177 patients with urea cycle disorders were evaluated regarding their condition1. Of the study population, a majority were diagnosed with OTC deficiency, accounting for 65% of confirmed diagnoses. Age of disease onset was categorized as neonatal (<29 days post-partum) or late (>29 days post-partum). The number of patients who experience late onset (n=80) was more than double that of neonatal onset (n=28), with most incidences occurring between 1 to 6 years of age1. The distribution of age of onset stratified by gender is presented in Figure 2 to the left1. Unfortunately, OTC deficiency is a potentially fatal disease that may result in death following the onset of symptoms. Within the study population, the 5-year survival rate was found to be 86% for the neonatal onset cohort and 92% for the late onset patients (FIgure 3)1. Kido et. al compared their findings to an earlier study published in 1998 and reported a twofold increase in the survival rate of late-onset patients at age 20 for both genders in 2012 compared to in 19981. The authors attributed this significant improvement to greater awareness of the disorder and the development of streamlined treatment protocols. Symptoms and Implications of OTC Deficiency In a healthy individual, basal ammonia levels are typically less than 65 µmol/L (180 µmol/L in neonates)3. However, urea cycle disorder patients often present with much higher levels of serum ammonia, which is one of the characteristic clinical signs of disorder. Patients may experience symptoms such as lethargy, vomiting and ataxia, with more severe cases resulting in seizures and coma3. Due to ammonia’s potency as a toxin to the nervous system, impaired cognitive functions are one of the primary long-term complications experienced by OTC deficiency patients1. In the study by Kido et al., blood ammonia level was found to be strongly correlated with the severity of disease outcome and cognitive disturbances1 (Figure 4). Of the patients examined with blood ammonia level between 60 to 180 μmol/L, 64% of the surviving patients did not develop mental retardation and exhibited normal EEG, CT and MRI patterns. 21% of the patients demonstrated abnormal brain patterns but had not expressed clinical signs of mental retardation, while the remaining 14% demonstrated mental retardation. In more severe cases in which ammonia levels exceed 360 μmol/L, there is a 15% observed death rate and a prevalence of mental retardation of 51%. The data suggests that the management of serum ammonia level is essential in mitigating the neurological implications of OTC deficiency1. Diagnosis One of the defining characteristics of urea cycle disorders, an elevated blood ammonia level, is often used as the first test in identifying affected individuals3. Those who exhibit serum ammonia levels above 100 µmol/L (>200 µmol/L in neonates) in the absence of other abnormal signs are likely experiencing a defect in the urea cycle3. Furthermore, plasma and urine amino acid profiles are also analyzed to allow for further tentative diagnosis. Those with OTCD will show increased levels of side products from the accumulation of substrates upstream of OTC in the urea cycle (eg. orotic acid from carbamoyl phosphate) and decreased levels of downstream metabolites (eg. citrulline and arginine). For a conclusive diagnosis of the type of urea cycle disorder, an enzymatic or genetic test is needed. OTC is only expressed in the liver, and thus necessitates a liver biopsy. Fortunately, recent advancements in genetics have allowed for the identification of OTC deficiency through gene sequencing, which is far less invasive. Structure and Mechanism of the OTC Enzyme Human OTC (EC 2.1.3.3) is a trimeric protein with three identical subunits. Each subunit is 354 amino acids in length, consisting of 14 alpha-helices, and 9 beta-sheets4 (Figure 5). The active site of OTC consists of 22 amino acid residues which catalyze the reaction between carbamoyl phosphate (CP) and ornithine (ORN) to form citrulline. The proposed mechanism for the catalytic activities of OTC involves a Sn2 displacement attack initiated by the amino group of the carbonyl carbon atom of the CP binding domain. Structurally, this domain is located near the interior of the protein trimer, and the ORN binding domains are located near the exterior4 (Figure 6). Mutations in OTC are among the most common inborn errors of the urea cycle and cause a deficiency in its catalytic effects, leading to various clinical manifestations. Many of these mutations are located near the active site and directly interfere with substrate binding (eg. K88N, R92Q, T93A)4. Other mutations lead to a variety of effects such as disruption of protein folding and protein destabilization (eg. S192Q, G195R, Q216E), interference with domain closure (eg. T178M, E181G), alteration of the charge of subunit surfaces to disrupt assembly of the trimer (eg. G79E, A102E, D126G), and interference with OTC’s interactions with other proteins (eg. R40H, Q180H, F354C)4. Locations of deleterious mutations which affect OTC are highlighted below in Figure 7. OTC Mutations and Effects A study by McCullough et al. attempted to characterize the relationship between genotype (the genetic defects present) and phenotype (how the patient presents) in those with OTCDs5. The study examined 157 families, collected tissue samples from patients ranging from neonates to adults, and identified 89 mutations5. ]] The common genetic mutations causing OTCD are displayed in Table 1 of the article, shown in Figure 8 to the left. This table displays current understandings of phenotypic classifications of patients with OTCD. Many mutations associated with neonatal-onset symptoms affect consensus splice site sequences, preventing the correct splicing of exons to form a functional OTC enzyme. In contrast, late-onset phenotypes often have missense mutations that do not affect splice site sequences. Almost all heterozygous females who present with hyperammonemia have neonatal-onset genotypes5. Interestingly, the age of symptom presentation varies from person to person. Newborns who completely lack OTC activity will present with symptoms at birth, and the disease almost always causes mortality within the first few days of life. However, patients with residual OTC activity vary in their age of presentation, with the oldest detected being 62 years old (Figure 9)5. Table II of the article, shown in Figure 10 to the right, presents further information regarding the age of onset for symptoms of various OTC mutations in males. The age of presentation varies greatly; in some cases, symptoms present as a child, and in other cases symptoms present as an adult (Figure 10). This strongly suggests that environmental factors, including exercise and diet, may influence the onset of OTCD symptoms in males presenting with a late-onset genotype5. Of interest are the peak ammonia levels in males presented with either neonatal or late-onset hyperammonemia shown in Figure 11. The median plasma ammonia levels are extremely different between the two groups, further implying that patients with late-onset symptoms have OTC mutations that allow residual activity of the enzyme5. Of particular interest is how males and females differ in their presentation of phenotypes. Figure 3 from the paper, shown in Figure 12, describes ammonia nitrogen incorporation into urea, and clearly identifies the differences between males and females. Males with a mutation that confer late-onset OTC deficiency all present with symptoms. In contrast, females typically appear asymptomatic if they contain an allele that would cause late-onset OTC deficiency symptoms in males. However, females presenting with symptoms were almost always heterozygotes with a neonatal-onset type mutation. These results indicate that females can often be asymptomatic, yet carry a mutation that would confer late-onset symptoms in males5. A similar study, conducted by Leibundgut et al. reports on several mutations found in clinical cases of OTC deficiency6. A mutation in arginine 141, which results in the introduction of a stop codon, is a common mutation based on the literature. It has been linked to a neonatal onset of disease in males and is characterized by undetectable OTC activity. The patient reported on in this study died one month after birth6. A H302Y mutant has been found to modify the structure of the ORN binding site of OTC. Similarly, there was no OTC activity detected in this patient, who fell into a comatose state on the second day of life and subsequently passed away on the 6th day. The authors hypothesized that drastic effects came about as a result of the substitution of the basic histidine residue for the neutral tyrosine residue6. The K88N and the P220A OTC mutants were shown to demonstrate partial residual OTC activity. The former patient had been healthy until the age of 4, but from then on, experienced episodic vomiting and lethargy. On the other hand, the latter patient suffered from protracted vomiting early on in life, fell into a comatose state at 2.5 years and eventually died as a result of cardiac insufficiency6. Finally, the K343T mutation introduced an additional positive charge to the protein structure, which was suspected to affect folding. However, the patient was reported to demonstrate residual enzyme activity, and as a result, it was hypothesized that this region of the enzyme was robust to modifications. Similar to other patients, the patient suffering from this mutation demonstrated repeated episodes of lethargy, as well as elevated blood ammonia levels and undetectable citrulline levels6. As shown, OTC deficiency displays varying clinical manifestations depending on the extent of impairment of enzymatic activity. However, elevations in orotic acid, a byproduct formed from carbamoyl phosphate as a result of an inability to metabolize carbamoyl phosphate through the urea cycle, is often seen in patients6. In another study by Qureshi et al., mouse models were used to elucidate the effects of OTC mutation on OTC enzyme kinetics and the urea cycle pathway7. The researchers bred female mice heterozygous for OTCD with unaffected male mice to yield 82 off-springs with 4 genotypes: normal males, normal females, hemizygous males, and heterozygous females. The progenies were enrolled in various enzymatic studies to measure the kinetics of OTC (Figure 13) as well as the excretion of urinary orotic acid and urea by each genotype (Figure 14). Figure 13 shows a significant decrease in the Km and Vmax''' of OTC based on concentrations of CP and ORN for mice with OTC deficiency. Vmax refers to the concentration of substrate at which the enzyme performs at its maximum rate and Km is Vmax divided by two. These results suggest that the mutation caused the enzyme to have an abnormal affinity for its two substrates, thus lowering the enzyme's maximal capacity for catalysis7. The results in Figure 14 demonstrate that the diseased mice exhibited increased levels of orotic acid which corresponds with the results of Leibundgut et. al mentioned previously. Additionally, the diseased mice have also shown decreased levels of urea, which was most likely due to the impairment of urea synthesis caused by the OTCD7. OTCD Treatments There is currently no cure for OTCD. In recent years, liver-targeted adenovirus gene transfer has been tested in clinical trials as a novel treatment option. However, limited efficacy and one fatal case involving systematic inflammatory response have halted its implementation8,9. Existing treatments focus on both limiting the production of nitrogenous wastes and maximizing the excretion of circulating ammonia. One of the most direct ways of controlling nitrogenous waste production is through the restriction of protein intake and shifting caloric intake to carbohydrates and lipids1. While it is ideal to minimize protein intake, intake of protein and essential amino acids is required to maintain nitrogen balance and decrease the breakdown of endogenous protein. Meanwhile, the rate of nitrogenous waste excretion can be improved with the administration of citrulline, sodium phenylbutyrate and sodium benzoate10. Citrulline is the product normally produced by OTC and used as is necessary for the incorporation of the second amino group in the urea cycle. The addition of citrulline allows the urea cycle to continue accepting amino group from aspartate before excreting it as urea and thus facilitates the management of nitrogenous waste. Sodium phenylbutyrate is not involved in the urea cycle but acts as a precursor in an alternative excretion pathway for nitrogen by binding free glutamine. Sodium benzoate treatment is perhaps the simplest in mechanism; it binds free amino acids for excretion and thus decreases serum ammonia levels. If management of urea cycle disorders through dietary restrictions is unsuccessful, further treatments by hemodialysis and peritoneal dialysis could be implemented to control the elevated levels of blood ammonia in events of hyperammonemic attack3. These treatments are able to achieve a rapid reduction in serum ammonia levels by direct filtration of the blood (hemodialysis) or diffusion through the peritoneum (peritoneal dialysis). Despite these efforts, some of the more severe cases may necessitate a liver-transplant. The Kido et al. study analyzes the effectiveness of hemodialysis in the event of hyperammonemic attacks1 (Figure 15). Of the patients who had blood ammonia level above 360 μmol/L (healthy: <65µmol/L ), 66% of the patients who exhibited no cognitive impairments were treated with hemodialysis. The study further analyzed the outcome of patients who received liver transplants and saw a great increase in their long-term survival rates. In comparison, liver transplants still offers the best long-term prognosis. The transplant group exhibited a perfect 5 year (post onset) and 94.1% 15 year (post onset) survival rate, compared to 89% and 81.5% for the non-transplant group respectively. Summary and Conclusion Based on the literature, it is evident that OTCDs are among the most common and devastating forms of urea cycle disorders. It has been found that there are a multitude of mutations that can arise in the OTC enzyme, resulting in altered levels of enzymatic activity as well as differing clinical manifestations. Overall, deficiencies in the urea cycle result in an accumulation of ammonia in body tissues, which is responsible for the most defining characteristics of OTCD symptoms such as cognitive impairments. Past literature has shown that the careful management of serum ammonia levels in OTCD patients could greatly alleviate the severity of the symptoms and improve long term outcome. Unfortunately, no current treatment options exist for OTCD and severe cases may require liver transplants as a last resort option. This highlights the importance of researching and refining novel treatment options such as retroviral gene therapy for OTCD patients. This review presents an overview of the literature regarding OTCDs and serves to further the understanding of the reader regarding all of aspects of the disease. It outlines the relevant clinical information regarding the disorder, which may be of interest to physicians and healthcare professions, as well as key structural and biochemical features of the enzyme, which may be used by biochemical researchers and students in the field. As well, the brochure provided summarizes the key information in a way that is easily understandable by patients and their families. While the body of literature has been impactful in providing information that is useful for clinicians, researchers and patients alike, there is still much that must be uncovered. First, Kido et. al provides an epidemiological study on the disorder in Japan, but it may be of interest to examine the disease in a Western or North American context, due to the possible effects of differing lifestyles and environments1. As was mentioned, there is currently no treatment option for those with OTCD and as a result, future research efforts must be aimed in this direction as well. Brochure for Patients Authors Biochem 3D03: Mike H, Adam S, Ryan D, Joshua X References (1) Kido, J.; Nakamura, K.; Mitsubuchi, H.; Ohura, T.; Takayanagi, M.; Matsuo, M.; Yoshino, M.; Shigematsu, Y.; Yorifuji, T.; Kasahara, M.; et al. Long-Term Outcome and Intervention of Urea Cycle Disorders in Japan. J. Inherit. Metab. Dis. '''2012, 35, 777–785. (2) Mitchell, S.; Ellingson, C.; Coyne, T.; Hall, L.; Neill, M.; Christian, N.; Higham, C.; Dobrowolski, S. F.; Tuchman, M.; Summar, M. Genetic Variation in the Urea Cycle: A Model Resource for Investigating Key Candidate Genes for Common Diseases. Human Mutation2008, 30, 56-60. (3) Leonard, J. V; Morris, A. A. M. Urea Cycle Disorders. Semin. Neonatol. 2002, 7'', 27–35. (4) Shi, D. 1.85-A Resolution Crystal Structure Of Human Ornithine Transcarbamoylase Complexed With N-Phosphonacetyl-L-Ornithine. CATALYTIC MECHANISM AND CORRELATION WITH INHERITED DEFICIENCY. ''Journal of Biological Chemistry 1998, 273, 34247-34254. (5) McCullough, B. A.; Yudkoff, M.; Batshaw, M. L.; Wilson, J. M.; Raper, S. E.; Tuchman, M. Genotype spectrum of ornithine transcarbamylase deficiency: correlation with the clinical and biochemical phenotype. Am. J. Med. Genet. '2000, 93'', 313-319. (6) Leibundgut, E. O.; Wermuth, B.; Colombo, J-P.; Liechti-Gallati, S. Ornithine Transcarbamylase Deficiency: Characterization of Gene Mutations and Polymorphisms ''Human Mutation ''1996', 8, 333-339 (7) Qureshi, I.; Letarte, J.; Ouellet, R. Ornithine Transcarbamylase Deficiency In Mutant Mice I. Studies On The Characterization Of Enzyme Defect And Suitability As Animal Model Of Human Disease.''Pediatr Res 1979, 13, 807-811. (8) Raper, S. E.; Yudkoff, M.; Chirmule, N.; Gao, G.-P.; Nunes, F.; Haskal, Z. J.; Furth, E. E.; Propert, K. J.; Robinson, M. B.; Magosin, S.;et al.''A Pilot Study of in Vivo Liver-Directed Gene Transfer with an Adenoviral Vector in Partial Ornithine Transcarbamylase Deficiency.''Hum. Gene Ther.2002,13, 163–175. (9) Raper, S. E.; Chirmule, N.; Lee, F. S.; Wivel, N. A.; Bagg, A.; Gao, G.; Wilson, J. M.; Batshaw, M. L. Fatal Systemic Inflammatory Response Syndrome in a Ornithine Transcarbamylase Deficient Patient Following Adenoviral Gene Transfer.Mol. Genet. Metab.2003,80, 148–158. (10) Maestri, N.; Brusilow, S.; Clissold, D.; Bassett, S. Long-Term Treatment of Girls with Ornithine Transcarbamylase Deficiency http://www.nejm.org.libaccess.lib.mcmaster.ca/doi/full/10.1056/nejm199609193351204#t=articleTop (accessed Nov 25, 2014).